Amino acid sequences capable of facilitating penetration across a biological barrier

ABSTRACT

The invention relates to amino acid sequences capable of facilitating penetration of an effector across a biological barrier. The invention also relates to methods of treating preventing diseases by administering or penetrating modules to affected subjects.

TECHNICAL FIELD OF THE INVENTION

This invention relates to amino acid sequences capable of facilitatingpenetration of an effector across biological barriers.

BACKGROUND OF THE INVENTION

Techniques enabling efficient transfer of a substance of interest acrossa biological barrier are of considerable interest in the field ofbiotechnology. For example, such techniques may be used for thetransport of a variety of different substances across a biologicalbarrier regulated by tight junctions (i.e., the mucosal epithelia, whichincludes the intestinal and respiratory epithelia and the vascularendothelia, which includes the blood-brain barrier).

The intestinal epithelium represents the major barrier to absorption oforally administered compounds, e.g., drugs and peptides, into thesystemic circulation. This barrier is composed of a single layer ofcolumnar epithelial cells (primarily enterocytes, goblet cells,endocrine cells, and paneth cells), which are joined at their apicalsurfaces by the tight junctions. See Madara et al., PHYSIOLOGY OF THEGASTROINTESTINAL TRACT; 2^(nd) Ed., Johnson, ed., Raven Press, New York,pp. 1251-66 (1987).

Compounds that are presented in the intestinal lumen can enter the bloodstream through active or facilitative transport, passive transcellulartransport, or passive paracellular transport. Active or facilitativetransport occurs via cellular carriers, and is limited to transport oflow molecular weight degradation products of complex molecules such asproteins and sugars, e.g., amino acids, pentoses, and hexoses. Passivetranscellular transport requires partitioning of the molecule throughboth the apical and basolateral membranes. This process is limited torelatively small hydrophobic compounds. See Jackson, PHYSIOLOGY OF THEGASTROINTESTINAL TRACT 2^(nd) Ed., Johnson, ed., Raven Press, New York,pp. 1597-1621 (1987). Consequently, with the exception of thosemolecules that are transported by active or facilitative mechanisms,absorption of larger, more hydrophilic molecules is, for the most part,limited to the paracellular pathway. However, the entry of moleculesthrough the paracellular pathway is primarily restricted by the presenceof the tight junctions. See Gumbiner, Am. J. Physiol., 253:C749-C758(1987); Madara, J. Clin. Invest., 83:1089-94 (1989).

In transmission electron microscopy, tight junctions appear as anapproximately 80 nm long region at the boundary of neighboring cells inwhich the plasma membranes of adjacent cells are brought into closeopposition. See Farquhar, et al., J. Cell Biol., 17:375-412 (1963). Thisstructure circumscribes epithelial cells immediately below the brushborder (apical domain), thereby forming a seal between epithelial cellsand their neighbors. See Pappenheimer, et al., J. Membrane Biol.,102:2125-2136 (1986); Claude et al., J. Cell Biol., 58:390-400 (1973);and Bakker, et al., J. Membrane Biol., 98:1209-1221 (1985). The tightjunctions function to define apical and basal membrane polarity bylimiting the exchange of membrane lipids and proteins between the twoand to regulate the paracellular transport of water, solutes, and immunecells. See Heiskala, et al., Traffic, 2:92-98 (2001).

Considerable attention has been directed to finding ways to increaseparacellular transport by “loosening” tight junctions. One approach toovercoming the restriction to paracellular transport is toco-administer, in a mixture, biologically active ingredients withabsorption enhancing agents. Generally, intestinal/respiratoryabsorption enhancers include, but are not limited to, calcium chelators,such as citrate and ethylenediamine tetraacetic acid (EDTA);surfactants, such as sodium dodecyl sulfate, bile salts,palmitoylcamitine, and sodium salts of fatty acids; or toxins, such aszonula occludens toxin (“ZOT”). ZOT functions by increasing thebioavailability of oral insulin in diabetic animals. See Fasano andUzzau, J. Clin. Invest., 99:1158-64 (1997). EDTA, which is known todisrupt tight junctions by chelating calcium, enhances the efficiency ofgene transfer into the airway respiratory epithelium in patients withcystic fibrosis. See Wang, et al., Am. J. Respir. Cell Mol. Biol.,22:129-138 (2000). However, one drawback to all of these methods is thatthey facilitate the indiscriminate penetration of any nearby moleculethat happens to be in the gastrointestinal or airway lumen. In addition,each of these intestinal/respiratory absorption enhancers has propertiesthat limit their general usefulness as a means to promote absorption ofvarious molecules across a biological barrier.

Moreover, with the use of surfactants, the potential lytic nature ofthese agents raises concerns regarding safety. Specifically, theintestinal and respiratory epithelia provides a barrier to the entry oftoxins, bacteria and viruses from the hostile exterior. Hence, thepossibility of exfoliation of the epithelium using surfactants, as wellas the potential complications arising from increased epithelial repair,raise safety concerns about the use of surfactants asintestinal/respiratory absorption enhancers.

When calcium chelators are used as intestinal/respiratory absorptionenhancers, Ca⁺² depletion does not act directly on the tight junction,but, rather, induces global changes in the cells, including disruptionof actin filaments, disruption of adherent junctions, diminished celladhesion, and activation of protein kinases. See Citi, J. Cell Biol.,117:169-178 (1992). Moreover, as typical calcium chelators only haveaccess to the mucosal surface, and luminal Ca⁺² concentration may vary,sufficient amounts of chelators generally cannot be administered tolower Ca⁺² levels to induce the opening of tight junctions in a rapid,reversible, and reproducible manner.

Additionally, some toxins such as Clostridium difficile toxin A and B,appear to irreversibly increase paracellular permeability and are thus,associated with destruction of the tight junction complex. See Hecht, etal., J. Clin. Invest., 82:1516-24 (1988); Fiorentini and Thelestam,Toxicon, 29:543-67 (1991). Other toxins such as Vibrio cholerae zonulaoccludens toxin (ZOT) modulate the structure of intercellular tightjunctions. As a result, the intestinal mucosa becomes more permeable.See Fasano, et al., Proc. Nat. Acad Sci., USA, 8:524246 (1991); U.S.Pat. No. 5,827,534. However, this also results in diarrhea.

Thus, a need remains for an efficient, specific, non-invasive, low-riskmeans to target various biological barriers for the delivery ofbiologically active molecules such as polypeptides, drugs and othertherapeutic agents.

SUMMARY OF THE INVENTION

The present invention provides penetrating peptides, which are capableof translocating across a biological barrier. The invention also relatesto methods of using such penetrating peptides to translocate an effectoracross a biological barrier.

In one aspect, the invention involves a penetrating peptide having atleast one amino acid sequence selected from: (BX)₄Z(BX)₂ZXB;ZBXB₂XBXB₂XBX₃BXB₂X₂B₂; ZBZX₂B₄XB₃ZXB₄Z₂B₂; ZB₉XBX₂B₂ZBXZBX₂;BZB₈XB₉X₂ZXB; B₂ZXZB₅XB₂XB₂X₂BZXB₂; XB₉XBXB₆X₃B; X₂B₃XB₄ZBXB₄XB_(n)XB;XB₂XZBXZB₂ZXBX₃BZXBX₃B; BZXBXZX₂B₄XBX₂B₂XB₄X₂; BZXBXZX₂B₄XBX₂B₂XB₄;B₂XZ₂XB₄XBX₂B₅X₂B₂; B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂;B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; X₃ZB₆XBX₃BZB₂X₂B₂; and at least12 contiguous amino acids of any of these amino acid sequences, where Xis any amino acid; B is a hydrophobic amino acid belonging to the groupconsisting of A, V, L, L M, F and P (single letter amino acid codes);and Z is a charged amino acid belonging to the group consisting of K, R,D and E; and where q is 0 or 1; m is 1 or 2; and n is 2 or 3; and wheret is 1 or 2 or 3; and where the penetrating peptide is capable oftranslocating across a biological barrier.

In one embodiment, the invention provides a penetrating peptide havingan amino acid sequence of any one of SEQ ID NOS: 1-15 and 24-29. Inaddition, the penetrating peptides of the invention include peptideshaving at least 12 contiguous amino acids of any of the peptides definedby SEQ ID NOS: 1-15 and 24-29. In various embodiments, the penetratingpeptide can be less than thirty (30), less than twenty-five (25), orless than twenty (20) amino acids in length. The invention also includesmutant or variant peptides any of whose residues may be changed from thecorresponding residues shown in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 24, 25, 26, 27, 28 or 29, while still encoding apeptide that maintains its penetrating activities and physiologicalfunctions, or functional fragments thereof In one embodiment, thefragment of an amino acid sequence of any one of SEQ ID NOS: 1-15 and24-29 is at least 10 amino acids in length. The amino acid sequence ofthe penetrating peptide variant may contain a conservative or anon-conservative amino acid substitution.

In general, a penetrating peptide variant that preserves thetranslocating function includes any variant in which residues at aparticular position in the sequence have been substituted by other aminoacids, and further includes the possibility of inserting an additionalresidue or residues between two residues of the parent protein as wellas the possibility of deleting one or more residues from the parentsequence. Any such amino acid substitution, insertion, or deletion isencompassed by the invention. In favorable circumstances, thesubstitution is a conservative substitution.

In some embodiments, amino acid substitutions at “non-essential” aminoacid residues can be made in the penetrating peptides. A “non-essential”amino acid residue is a residue that can be altered from the nativesequences of the penetrating peptides without altering their biologicalactivity, whereas an “essential” amino acid residue is required for suchbiological activity. For example, amino acid residues that are conservedamong the penetrating peptides of the invention are predicted to beparticularly non-amenable to substantial alteration. Amino acids forwhich conservative substitutions can be made are well known within theart.

Mutations can be introduced into nucleic acids encoding penetratingpeptides by standard techniques, including, but not limited tosite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predicted,non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined within the art.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted non-essentialamino acid residue in the penetrating peptide is replaced with anotheramino acid residue from the same side chain family. Alternatively, inanother embodiment, mutations can be introduced randomly along all orpart of a penetrating peptide coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity. Followingmutagenesis, the encoded penetrating peptide can be expressed by anyrecombinant technology known in the art and the activity of the proteincan be determined. Amino acid substitutions can also be introducedduring artificial peptide synthesis such as solid-phase synthesis ofpeptides.

The relatedness of amino acid families may also be determined based onside chain interactions. Substituted amino acids may be fully conserved“strong” residues or fully conserved “weak” residues. The “strong” groupof conserved amino acid residues may be any one of the following groups:STA, NREQK, NHQK, NDEQ, QHRK, MILV, MILF, HY, FYW, wherein the singleletter amino acid codes are grouped by those amino acids that may besubstituted for each other. Likewise, the “weak” group of conservedresidues may be any one of the following: CSA, ATV, SAG, STNK, STPA,SGND, SNDEQK, NDEQHK, NEQHRK, HFY, wherein the letters within each grouprepresent the single letter amino acid code.

The invention also includes analogs in which one or more peptide bondshave been replaced with an alternative type of covalent bond (a “peptidemimetic”) that is not susceptible to cleavage by peptidases elaboratedby the subject. Where proteolytic degradation of a peptide compositionis encountered following administration to the subject, replacement of aparticularly sensitive peptide bond with a noncleavable peptide mimeticrenders the resulting peptide derivative compound more stable and thusmore useful as a therapeutic. Such mimetics, and methods ofincorporating them into peptides, are well known in the art.

Similarly, the replacement of an L-amino acid residue by a D-amino acidresidue is a standard method for rendering the compound less sensitiveto enzymatic destruction. Other amino acid analogs are known in the art,such as norleucine, norvaline, homocysteine, homoserine, ethionine, andthe like. Also useful is derivatizing the compound with anamino-terminal blocking group such as a t-butyloxycarbonyl, acetyl,methyl, succinyl, methoxysuccinyl, suberyl, adipyl azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyaselayl,methoxyadipyl, methoxysuberyl, and a 2,3-dinitrophenyl group.

The invention also includes the replacement of the N-terminal amino acidin said sequences by methionine or the addition of between 1 to 4 aminoacids, starting with methionine, at the N-terminus of the sequences inorder to enable the biosynthesis of recombinant constructs of thesequences using the canonical start codon of methionine, which iswell-known in the art.

In some embodiments, the penetrating peptide of the invention can befurther chemically modified. For example, one or more polyethyleneglycol (PEG) residues can be attached to the penetrating peptides of theinvention.

As used herein, a “penetrating peptide” is any peptide that facilitatesthe translocation of a substance across a biological barrier. Examplesof biological barriers include, but are not limited to, tight junctionsand the plasma membrane. Moreover, those skilled in the art willrecognize that translocation may occur across a biological barrier in atissue such as epithelial cells or endothelial cells.

In some embodiments, the invention involves a penetrating module that isa penetrating peptide of the invention fused, coupled, or attached to aneffector. The “effector” can be any suitable molecule, including, butnot limited to DNA, RNA, a protein, a peptide, or a pharmaceuticallyactive agent, such as, for example, a hormone, a growth factor, aneurotrophic factor, a bioactive peptide, heparin, a toxin, anantibiotic, an antipathogenic agent, an antigen, an antibody, anantibody fragment, an immunomodulator, and an enzyme; or a therapeuticagent. Bioactive peptides include, for example, insulin, growth hormone,a gonadotropin, a growth factor, erythropoietin, granulocyte/monocytecolony stimulating factor (GM-CSF), αMSH, enkephalin, dalargin,kyotorphin, EPO, bFGF, hirulog, a lutenizing hormone releasing hormone(LHRH) analog, and neurotrophic factors. Pharmaceutically active agentsinclude, for example, an anticoagulant, a toxin, an antibiotic, anantipathogenic agent, an antigen, an antibody fragment, a vitamin, animmunomodulator, an enzyme, an antineoplastic agent, heparin,methotraxate, and a therapeutic agent.

As used herein, the terms “fusion” or “fused” or “coupled” or “attached”are meant to include all such specific interactions that result in twoor more molecules showing a preference for one another relative to somethird molecule including any type of interaction enabling a physicalassociation between an effector or a molecular vessel and a penetratingpeptide. This includes, but is not limited to, processes such ascovalent, ionic, hydrophobic, and hydrogen bonding, but does not includenon-specific associations such as solvent preferences. The associationmust be sufficiently strong so that the effector does not disassociatebefore or during penetration of the biological barrier. Fusion may beachieved using any chemical, biochemical, enzymatic or genetic couplingmethod known to those skilled in the art. The effector of interest ispreferably coupled to the C-terminal end of the penetrating peptide.

In other embodiments, the penetrating peptide could also be a complexconsisting of the penetrating peptide attached to a “molecular vessel”in which an effector is enclosed. Suitable effectors include, but arenot limited to, any of the suitable bioactive peptides orpharmaceutically active agents described herein. A molecular vesselcould be a soluble receptor, or part of a soluble receptor (e.g., a“minireceptor”, as described in Kristensens, et al., J. Biol. Chem.,274(52):37351-56 (1999)). A molecular vessel can also be a bindingprotein. The molecular vessel will serve as a high affinity bindingpocket for the delivery of the intact effector, such as a hormone, agrowth factor, or any other effector. One example of a molecular vesselis a soluble insulin receptor, or the ligand-binding domain of theinsulin receptor (e.g., the above-mentioned “minireceptor”), to bind anddeliver insulin as an effector. Another example for the use of amolecular vessel to deliver non-permeable effectors, is the use ofIntrinsic factor, attached to the penetrating peptide, in order toenable the delivery of vitamin B12 as an effector. In a way, themolecular vessel serves as a “Trojan horse” to translocate the otherwiseimpermeable effector across a biological barrier. The “effector” can beany suitable molecule as defined above.

Another embodiment of the invention involves a method of bulktranslocation of a pharmaceutical composition. The peptides describedherein serve as the basis for the design of therapeutic drug-containingmicro particles/droplets. For example, penetrating peptides are attachedto fatty moieties and incorporated at the interface of a hydrophobicvesicle which contains the desired pharmaceutical composition. This canbe achieved via amidation of the free amino group of extra lysine(s),added at the C-terminus of the penetrating peptide, using long fattyacids such as stearoyl, palmitoyl or myristoyl.

Another embodiment of the invention involves a method of oralvaccination by administering to a subject a penetrating peptide of theinvention attached to a desirable antigenic protein.

In one embodiment, the invention involves a method of translocating apenetrating peptide of the invention across a biological barrier. Inanother embodiment, the invention involves methods of translocating aneffector across a biological barrier by coupling the effector to apenetrating peptide module that can be introduced to the biologicalbarrier.

As used herein, the term “biological barrier” is meant to includebiological membranes such as the plasma membrane as well as anybiological structures sealed by tight junctions (or occluding junctions)such as the mucosal epithelia, including, but not limited to, theintestinal or respiratory epithelia or the vascular endothelia,including, but not limited to, the blood-brain barrier.

In still further embodiments, the invention includes a pharmaceuticalcomposition containing a therapeutically or prophylactically effectiveamount of one or more penetrating peptide and a pharmaceuticallyacceptable carrier.

Preferred “pharmaceutical compositions” include enteric coated tabletsand gelatin capsules comprising the active ingredient together with a)diluents, e.g. lactose, dextrose, sucrose, mannitol, sorbitol, celluloseand/or glycine; b) protease inhibitors such as Aprotinin or trasylol; c)lubricants, e.g., silica, talcum, stearic acid, its magnesium or calciumsalt, poloxamer and/or polyethyleneglycol; for tablets also d) binders,e.g., magnesium aluminum silicate, starch paste, gelatin, tragacanth,methylcellulose, sodium carboxymethylcellulose and/orpolyvinylpyrrolidone; e) ionic surface active agents such as bile salts,if desired f) disintegrants, e.g., starches, agar, alginic acid or itssodium salt, or effervescent mixtures; and/or g) absorbents, colorants,flavors and sweeteners. Injectable compositions are preferably aqueousisotonic solutions or suspensions, and suppositories are advantageouslyprepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, reducing agentse.g., NAC (N-Acetyl-L-Cysteine), stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. The compositions are prepared according toconventional mixing, granulating or coating methods, respectively, andcontain about 0.01 to 75%, preferably about 0.1 to 10%, of the activeingredient.

These compositions may further contain a mixture of at least twosubstances selected from the group consisting of a non-ionic detergent,an ionic detergent, a protease inhibitor, and a reducing agent. Forexample, the non-ionic detergent may be a poloxamer; the poloxamer maybe pluronic F-68; the ionic detergent may be a bile salt; and the bilesalt may be Taurodeoxychilate; the protease inhibitor may be selectedfrom the group consisting of aprotonir and soy bean trypsin inhibitor,and/or the reducing agent may be NAC.

The invention also provides a method for producing a penetrating peptideby coupling an effector to a penetrating peptide. For example, thepenetrating peptides can be produced by transfecting a production cellwith a vector that has a nucleic acid molecule of a fusion proteinencoding the penetrating peptide and an effector operably linked to anexpression control sequence; culturing the production cell underconditions that permit production of a fusion protein that includes thepenetrating peptide and an effector peptide; and isolating the fusionprotein. For example, the penetrating peptides can also be produced byusing solid-phase peptides synthesis methods, as is well known in theart. Penetrating peptides that are produced by the methods for producinga penetrating peptide of the invention can also be further chemicallymodified. For example, one or more polyethylene glycol (PEG) residuescan be attached to the penetrating peptides of the invention.

In another embodiment, the invention provides a method of producing apenetrating peptide comprising a penetration peptide and an effector.The effector may be coupled to the penetration peptide by a covalent ora non-covalent bond. For example, the covalent bond may be a peptidebond or the covalent bond may be achieved by a homo- or ahetero-functional bridging reagent. The bridging reagent may be asuccinimidyl-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC)-typereagent. The covalent bond may also be achieved by using a peptidelinker. In some embodiments, the peptide linker may have an amino acidsequence of SEQ ID NO: 16 or 17 and may be cleaved by an enzyme. In someembodiments, the enzyme may be conditionally activated under a certainphysiological state, and the released factor may favorably influencethat physiological state. In other embodiments, the non-covalent bondmay be achieved by an attachment of a hydrophobic moiety to thepenetrating peptide, such that the hydrophobic moiety enables thepenetrating peptide to be incorporated at the interface of a hydrophobicvesicle in which the effector is contained. In other embodiments, thenon-covalent bond may be the result of a biotin-avidin orbiotin-streptavidin interaction.

In various other embodiments, the penetrating peptides are derived froma pathogenic or non-pathogenic bacteria, characterized by the ability topenetrate biological barriers in vivo. In some embodiments, thepenetrating peptides are derived from integral membrane proteins ofpathogenic or non-pathogenic bacteria. In other embodiments, thepenetrating peptides are derived from extracellular proteins released bypathogenic or non-pathogenic bacteria. In other embodiments, thepenetrating peptide is derived from a bacterial toxin. In still otherembodiments, the penetrating peptides are derived from human neurokininreceptors, characterized by the ability to penetrate biological barriersin viva, or are derived from viral proteins.

In still further embodiments, the invention includes a kit having one ormore containers containing a therapeutically or prophylacticallyeffective amount of a pharmaceutical composition.

In still further embodiments the concept of a conditionally-activatedeffector is designed to allow selective release and activation ofproteins under conditions where and at sites in which their activity isbeneficial.

In this embodiment, the beneficial protein (the effector) is coupled tothe penetrating peptide through a cleavable linker peptide. The cleavagesite is endogenously recognized and cleaved by an enzyme of the cascadetargeted by the effector. The released effector may act as an inhibitoryprotein or an activator of desired processes.

Finally, another embodiment of the invention involves a method oftreating or preventing a disease or pathological condition byadministering to a subject in which such treatment or prevention isdesired, a pharmaceutical composition in an amount sufficient to treator prevent the disease or pathological condition. For example, thedisease or condition to be treated may include but are not limited toendocrine disorders, including diabetes, infertility, hormonedeficiencies and osteoporosis; neurodegenerative disorders, includingAlzheimer's disease, Parkinson's disease, and Huntington's disease;cardiovascular disorders, including atherosclerosis, hyper- andhypocoagulable states, coronary disease, and cerebrovascular events;metabolic disorders, including obesity and vitamin deficiencies;haematological disorders; and neoplastic disease.

The details of one or more embodiments of the invention have been setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequence alignment of ORF HI0638 and itshomologs from other pathogenic bacteria

FIG. 2 shows amino acid sequence alignment of the peptides used in thisinvention as penetrating modules as well as their organism of origin.

DETAILED DESCRIPTION OF THE INVENTION

The advantages of the use of small peptide carriers include high qualityand purity, low immunogenicity and the potential for highly efficientdelivery to biological barriers in an organism. Accordingly, peptidecarriers have the potential to improve upon conventional transporterssuch as liposomes or viruses for the efficient delivery of manymacromolecules. The present invention employs a short peptide motif tocreate a penetrating module to specifically transport macromoleculesacross biological barriers sealed by tight junctions.

The present invention provides a peptide penetration system thatspecifically targets various tissues, especially epithelial andendothelial ones, for the delivery of drugs and other therapeutic agentsacross a biological barrier. Existing transport systems in the art aretoo limited to be of general application because they are inefficient,alter the biological properties of the active substance, kill the targetcell, irreversibly destroy the biological barrier and/or pose too highof a risk to be used in human subjects.

The peptide penetration system of the present invention uses conservedpeptide sequences from various proteins involved in paracytosis tocreate a penetrating module capable of crossing biological barriers. Forexample, a peptide encoded by or derived from ORF HI0638 of Haemophilusinfluenzae facilitates penetration of this bacterium between human lungepithelial cells without compromising the epithelial barrier. Thepeptide sequence encoded by ORF HI0638 is conserved in common pathogenicbacteria or symbiotic including, for example, Haemophilus influenzae,Pasteurella multocida, Escherichia coli, Vibrio cholerae, Buchneraaphidicola, Pseudomonas aeruginosa, and Xylella fastidiosa. A peptidehomologous to the N-terminal sequence of HI0638 is also found in otherbacteria including, for example, Rhizobium loti, Chlamydia pneumoniae,NprB from Bacillus subtilis, and pilins from Kingella dentrificans andEikenella corrodens.

Furthermore, a similar peptide sequence is also conserved in proteins ofeukaryotic origin such as the neurokinin receptor family proteins,including the human NK-1 and NK-2 receptors. It is known that theneurokinin receptor family is involved in the control of intercellularpermeability including plasma extravasation and oedema formation.Extravasation, the leakage and spread of blood or fluid from vesselsinto the surrounding tissues, often follows inflammatory processesinvolved in tissue injury, allergy, burns and inflammation. Inparticular, when NK-1 receptors on blood vessels are activated skininflammation occurs due to an increase in vascular permeability. SeeInoue, et al., Inflamm. Res., 45:316-323 (1996). The neurokinin NK-1receptor also mediates dural and extracranial plasma proteinextravasation, thereby implicating the NK-1 receptor in thepathophysiology of migraine headache. See O'Shaughnessy and Connor,Euro. J. of Pharm., 236:319-321 (1993). The sequences of examplepenetrating peptides of the invention are shown in Table 1. TABLE 1Peptide/Organism Sequence SEQ ID NO Peptide 1: from ORF HI0638NYHDIVLALAGVCQSAKLVHQLA (SEQ ID NO:1) Haemophilus influenzae Peptide 2:from PM1850 NYYDITLALAGVCQAAKLVQQFA (SEQ ID NO:2) Paasteurella multocidaPeptide 3: from YCFC NYYDITLALAGICQSARLVQQLA (SEQ ID NO:3) Escherichiacoli Peptide 4: from VC1127 AIYDRTIAFAGICQAVAIVQQVA (SEQ ID NO:4) Vibriocholerae Peptide 5: from BU262 KIHLITLSLAGICQSAHLVQQLA (SEQ ID NO:5)Buchnera aphidicola Peptide 6: from PA2627 DPRQQLIALGAVFESAALVDKLA (SEQID NO:6) Pseudomonas aeruginosa Peptide 7: from XF1439LIDNRVLALAGVVQALQQVRQIA (SEQ ID NO:7) Xylella fastidiosa Peptide 8: fromMLR0187 NLPPIVLAVIGICAAVFLLQQYV (SEQ ID NO:8) Rhizobium loti Peptide 9:from Human NK-2 NYFIVNLALADLCMAAFNAAFNF (SEQ ID NO:9) Receptor Peptide10: from CPN0710/C TAFDFNKMLDGVCTYVKGVQQYL (SEQ ID NO:10) Chlamydiapneumoniae Peptide 11: from MLR4119 RAILIPLALAGLCQVARAGDISS (SEQ IDNO:11) Rhizobium loti Peptide 12: from NprB MRNLTKTSLLLAGLCTAAQMVFVTH(SEQ ID NO:12) Bacillus subtilis Peptide 13: from PilinIELMIVIAIIGILAAIALPAYQEYV (SEQ ID NO:13) Kingella dentrificans Peptide14: from Pilin IELMIVIAIIGILAAIALPAYQDYV (SEQ ID NO:14) Eikenellacorrodens Peptide 15: from zonula ASFGFCIGRLCVQDGF (SEQ ID NO:15)occludens toxin (ZOT) Peptide 29: from Human NK-1NYFLVNLAFAEASMAAFNTVVNF (SEQ ID NO:24) Receptor Peptide 30: from YCFCMNYYDITLALAGICQSARLVQQLA (SEQ ID NO:25) Escherichia coli Peptide 31:from YCFC MYYDITLALAGICQSARLVQQLA (SEQ ID NO:26) Escherichia coilPeptide 32: from YCFC MYDITLALAGICQSARLVQQLA (SEQ ID NO:27) Escherichiacoil Peptide 33: from NprB MRNLTRTSLLLAGLCTAAQMVFV (SEQ ID NO:28)Bacillus subtilis Peptide 34: from ORF HI0638 NYHDIVLALAGVCQSARLVHQLA(SEQ ID NO:29) Haemophilus influenrae

The penetrating peptides of the instant invention also include peptidescontaining at least 12 contiguous amino acids of any of the peptidesdefined by SEQ ID NOS: 1-15 and 24-29.

The peptide penetration system of the present invention exhibitsefficient, non-invasive delivery of an unaltered biologically activesubstance. For example, the penetrating peptides of the invention can beused in the treatment of diabetes. Insulin levels in the blood streammust be tightly regulated. The penetrating modules of the invention canbe used to deliver insulin across the mucosal epithelia at high yield.Alternative non-invasive insulin delivery methods, previously known inthe art, have typical yields of 1-5% and cause intolerable fluctuationsin the amount of insulin absorbed.

In addition, these penetrating modules or peptides also can be used totreat conditions resulting from atherosclerosis and the formation ofthrombi and emboli such as myocardial infarction and cerebrovascularaccidents. Specifically, the penetrating modules can be used to deliverheparin across the mucosal epithelia.

The penetrating modules or peptides of the invention also can be used totreat female fertility problems. For example, the penetrating modules orpeptides can be used to transport follicle stimulating hormone (“FSH”)across the mucosal epithelia.

Previously, the delivery of effectors (e.g., the delivery of insulin orheparin to the blood stream) required invasive techniques such asintravenous or intramuscular injections. One advantage of thepenetrating modules or peptides is that they can deliver effectorsacross biological barriers through non-invasive administration,including, for example oral, bucal, inhalation, insufflation,transdermal, or depository. In addition, a further advantage of thepenetrating modules of the invention is that they can cross theblood-brain barrier, thereby delivering effectors to the CNS.

The peptides described herein serve as the basis for the design oftherapeutic “cargos”, namely the coupling of the carriers (“penetratingpeptide”) with one or more therapeutic agents (“effectors”). A covalentor noncovalent bond can be used to couple a penetrating peptide to oneor more effectors or molecular vessels. Peptide linkers that are cleavedby endogenous peptidases can be used to couple penetrating peptides andeffectors or molecular vessels. One example of a detachable peptidelinker is the amino acid sequence IEGR (SEQ ID NO: 16) that can becleaved in the bloodstream by factor Xa (see, e.g., Karlheinz, P., etal., Circulation 101:1158-1164 (2000)).

Peptide linkers are short sequences appearing between the amino acidsequences of the penetrating peptide and the effector. These peptidelinkers are cleaved by endogenous peptidases or other enzymes, therebydissociating the coupling of penetrating peptide to the effector. Suchpeptide linkers can be utilized for the creation of conditionallyactivated effectors. Conditionally activated effectors are coupled topenetrating peptides by peptide linkers that are preferentially cleavedduring specific physiological processes. Once cleaved, the effector isreleased to modulate the process—for instance, inhibition of a signalcascade. Preferential cleavage is achieved by using linker sequencescleaved by proteases that are physiologically activated by the triggeredcascade. Thus, the conditionally activated effector serves as a“negative feedback” mechanism that inhibits the physiologic processwhich activated it.

One example of a conditionally activated effector is a thrombolysisactivator, e.g., tPA (tissue plasminogen activator). By using the abovementioned linker (i.e., SEQ ID NO. 16) for cleavage by factor Xa, tPA ispreferentially released in sites of active coagulation. This wayexcessive coagulation cascades can be modulated to reduce pathologicthrombosis due to the local release of tPA. Alternatively, othercoagulation inhibitors may be used in place of tPA, e.g., hirudin,hirolog, protein C, protein C activator or protein S.

Another example of conditionally activated effectors is a complementcascade inhibitor, e.g., Clq inhibitor. In this example a peptide linkeris incorporated that is specifically cleaved by elements of thecomplement cascade, e.g., C3a Thus, excessive complement activation isinhibited, thereby attenuating the extent of pathologic inflammatoryprocesses.

By way of non-limiting example, conditionally activated effectorsaccording to the invention may be used, e.g., in the inhibition ofexcessive blood clotting, in the inhibition of pathological inflammatoryprocesses, in the inhibition of high blood pressure and congestive heartfailure by angiotensin converting enzyme (ACE)-dependent release of anappropriate effector such as brain-derived natriuric peptide (BNP), orin the inhibition of excessive extracellular-matrix degradation, such asthe degradation that occurs during inflammatory processes or tumorinvasion, by matrix metaloproteinases (MPP)-dependent release of aneffector such as tissue inhibitor of metaloproteinases (TIMP).

Alternatively, the penetrating peptide can be attached to a linker towhich imaging compounds can be covalently attached, for example throughfree amino groups of lysine residues. Such a linker includes, but is notlimited to, the amino acid sequence GGKGGK (SEQ ID NO:17).

A penetrating peptide is a peptide that facilitates the passage,translocation, or penetration of a substance across a biologicalbarrier, particularly between cells “sealed” by tight junctions.Translocation may be detected by any method known to those skilled inthe art, including using radioactive tagging and/or fluorescent probesincorporated into a penetrating module in conjunction with a paracytosisassay as described in, for example, Schilfgaarde, et al., Infect. andImmun., 68(8):4616-23 (2000). Generally, a paracytosis assay isperformed by: a) incubating a cell layer with a penetrating peptide ormodule; b) making cross sections of the cell layers; and c) detectingthe presence of the peptides or peptide modules. The detection step maybe carried out by incubating the fixed cell sections with labeledantibodies directed to the peptide, followed by detection of animmunological reaction between the peptide and the labeled antibody.Alternatively, the peptide may be labeled using a radioactive label, ora fluorescent label, or a dye in order to directly detect the presenceof the peptide. Further, a bioassay can be used to monitor the peptidetranslocation. For example, using a bioactive peptide such as insulin,attached to a penetrating module, the drop in blood glucose level can bemeasured.

As used herein, the term “effector” refers to any molecule or compoundof, for example, biological, therapeutic, pharmaceutical, diagnostic,tracing, or food processing interest. It may consist of nucleic acids(ribonucleic acid, deoxyribonucleic acid) from various origins, andparticularly of human, viral, animal, eukaryotic or prokaryotic, plant,synthetic origin, etc. A nucleic acid of interest may be of a variety ofsizes, ranging from, for example, a simple trace nucleotide to a genomefragment, or an entire genome. It may be a viral genome or a plasmid.Alternatively, the effector of interest may also be a protein, such as,for example, an enzyme, a hormone, a cytokine, an apolipoprotein, agrowth factor, a bioactive peptide, an antigen, or an antibody, etc.Furthermore, the effector may be a pharmaceutically active agent, suchas, for example, a toxin, a therapeutic agent, or an antipathogenicagent, such as an antibiotic, an antiviral, an antifungal, or ananti-parasitic agent. The effector can also be a physiologically activemolecule like a vitamin. The effector of interest may itself be directlyactive or may be activated in situ by the peptide, by the molecularvessel, by a distinct substance, or by environmental conditions.

The terms “pharmaceutically active agent” and “therapeutic agent” areused herein to refer to a chemical material or compound which, whenadministered to an organism, induces a detectable pharmacologic and/orphysiologic effect.

The penetrating peptide conjugates according to the present inventionare characterized by the fact that their penetration capacity isvirtually independent of the nature of the effector or molecular vesselthat is coupled to it.

Also included in the invention are methods of producing the penetratingpeptides and modules described herein. For example, a penetratingpeptide or module of the invention can be produced by standardrecombinant DNA techniques known in the art. By way of non-limitingexample, DNA fragments coding for the different polypeptide sequencescan be ligated together in-frame in accordance with conventionaltechniques, e.g., by employing blunt-ended or stagger-ended termini forligation, restriction enzyme digestion to provide for appropriateternini, filling-in of cohesive ends as appropriate, alkalinephosphatase treatment to avoid undesirable joining, and enzymaticligation. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.

Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers that give rise to complementary overhangs betweentwo consecutive gene fragments that can subsequently be annealed andreamplified to generate a chimeric gene sequence (see, e.g., Ausubel, elal. (eds.) CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,1992). Moreover, many expression vectors are commercially available thatalready encode a fusion moiety (e.g., a GST polypeptide). A penetratingpeptide-encoding nucleic acid can be cloned into such an expressionvector such that the fusion moiety is linked in-frame to the penetratingpeptide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

Recombinant expression vectors comprise a nucleic acid in a formsuitable for expression of the nucleic acid in a host cell, which meansthat the recombinant expression vectors include one or more regulatorysequences, selected on the basis of the host cells to be used forexpression, that is operatively-linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably-linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Expression vectors can be introduced into host cells to therebyproduce proteins or peptides, including fusion proteins or peptides,encoded by nucleic acids as described herein (e.g., penetrating peptidesor modules).

Recombinant expression vectors can be designed for expression ofpenetrating peptides or modules in prokaryotic or eukaryotic cells. Forexample, penetrating peptides or modules can be expressed in bacterialcells such as Escherichia coli, insect cells (using baculovirusexpression vectors), yeast cells or mammalian cells. Suitable host cellsare discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS INENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively,the recombinant expression vector can be transcribed and translated invitro, for example using T7 promoter regulatory sequences and T7polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant protein to enable separation of therecombinant protein from the fusion moiety subsequent to purification ofthe fusion protein. Such enzymes, and their cognate recognitionsequences, include Factor Xa, thrombin and enterokinase. Typical fusionexpression vectors include pGEX (Pharmacia Biotech Inc; Smith andJohnson, 1988. Gene 67: 3140), pMAL (New England Biolabs, Beverly,Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant protein.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET IId(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences encoding the penetrating peptides or modules of the inventioncan be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast Saccharomycescerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234),pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz etal., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, a penetrating peptide or module can be expressed ininsect cells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., SF9cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3:2156-2165) and the pVL series (Lucklow and Surmmers, 1989. Virology 170:31-39).

In yet another embodiment, a nucleic acid encoding the penetratingpeptides and modules of the invention is expressed in mammalian cellsusing a mammalian expression vector. Examples of mammalian expressionvectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman,et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, theexpression vector's control functions are often provided by viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. Forother suitable expression systems for both prokaryotic and eukaryoticcells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilarnent promoter; Byme and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the a-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule encoding the penetrating peptides and modulesof the invention cloned into the expression vector in an antisenseorientation. That is, the DNA molecule is operatively-linked to aregulatory sequence in a manner that allows for expression (bytranscription of the DNA molecule) of an RNA molecule that is antisenseto the penetrating peptide or module mRNA. Regulatory sequencesoperatively linked to a nucleic acid cloned in the antisense orientationcan be chosen that direct the continuous expression of the antisense RNAmolecule in a variety of cell types, for instance viral promoters and/orenhancers, or regulatory sequences can be chosen that directconstitutive, tissue specific or cell type specific expression ofantisense RNA. The antisense expression vector can be in the form of arecombinant plasmid, phagemid or attenuated virus in which antisensenucleic acids are produced under the control of a high efficiencyregulatory region, the activity of which can be determined by the celltype into which the vector is introduced. For a discussion of theregulation of gene expression using antisense genes see, e.g.,Weintraub, et aL, “Antisense RNA as a molecular tool for geneticanalysis,” Reviews-Trends in Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector has been introduced. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut also to the progeny or potential progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, thepenetrating peptide or module can be expressed in bacterial cells suchas E. coli, insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “nsfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding the penetrating peptide or module, or can be introduced on aseparate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (eg., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

A host cell, such as a prokaryotic or eukaryotic host cell in culture,can be used to produce (i.e., express) a penetrating peptide or module.Accordingly, the invention further provides methods for producingpenetrating peptides or modules using the host cells. In one embodiment,the method comprises culturing the host cell (into which a recombinantexpression vector encoding a penetrating peptide or module has beenintroduced) in a suitable medium such that the penetrating peptide ormodule is produced. In another embodiment, the method further comprisesisolating the penetrating peptide or module from the medium or the hostcell.

The penetrating peptides and modules of the invention can also beproduced using solid-phase peptide synthesis methods known in the art.For example, a penetrating peptide can be synthesized using theMerrifield solid-phase synthesis method. (See e.g., Merrifield, R. B.,J. Am. Chem. Soc. 85:2149 (1963); ENCYCLOPEDIA OF MOLECULAR BIOLOGY 806(1st ed. 1994). In this method, the C-terminal amino acid is attached toan insoluble polymeric support resin (e.g., polystyrene beads), therebyforming an immobilized amino acid. To avoid unwanted reactions as theC-terminal amino acid is attached to the resin, the amino group of theC-terminal amino acid is protected or “blocked” using, for example, atert-butyloxylcarbonyl (t-BOC) group. The blocking group, e.g., t-BOC,on the immobilized amino acid is then removed by adding a dilute acid tothe solution. Before a second amino acid is attached to the immobilizedpeptide chain, the amino-group of the second amino acid is blocked, asdescribed above, and the α-carboxyl group of the second amino acid isactivated through a reaction with dicyclohxylcarbdiimide (DCC). Theactivated a-carboxyl group of the second amino acid then reacts with thefree amino group of the immobilized amino acid to form a peptide bond.Additional amino acids are then individually added to the terminal aminoacid of the immobilized peptide chain according to the required sequencefor the desired penetrating peptide or module. Once the amino acids havebeen added in the required sequence, the completed peptide is releasedfrom the resin, such as for example, by using hydrogen fluoride, whichdoes not attack the peptide bonds.

The penetrating peptides or modules of the invention can also besynthesized using Fmoc solid-phase peptide synthesis. (See e.g.,University of Illinois at Urbana-Champaign Protein Sciences Facility,Solid-Phase Peptide Synthesis (SPPS), athttp://www.biotech.uiuc.edu/spps.htrn). In this method, the C-terminalamino acid is attached to an insoluble polymeric support resin (e.g.,polystyrene beads, cross-linked polystyrene resins, etc.), such as forexample, via an acid labile bond with a linker molecule. To avoidunwanted reactions as the C-terminal amino acid is being attached to theresin, the amino group of the C-terminal amino acid is blocked using anFmoc group. The blocking group, e.g., Fmoc, on the terminal amino acidof the immobilized amino acid is then removed by adding a base to thesolution. Side chain functional groups are also protected using anybase-stable, acid-labile groups to avoid unwanted reactions. Before thesecond amino acid is attached to the immobilized amino acid, theamino-group of the second amino acid is blocked, as described above, andthe α-carboxyl group of each successive amino acid is activated bycreating an N-hydrobenzotriazole (HOBt) ester in situ. The activatedα-carboxyl group of the second amino acid and the free amino group ofthe immobilized amino acid then react, in the presence of a base, toform a new peptide bond. Additional amino acids are then successivelyadded to the terminal amino acid of the immobilized peptide chain, untilthe desired peptide has been assembled. Once the necessary amino acidshave been attached, the peptide chain can be cleaved from the resin,such as for example, by using a mixture of trifluoroacetic acid (TFA)and scavengers (e.g., phenol, thioanisol, water, ethanedithiol (EDT) andtriisopropylsilan (TIS)) that are effective to neutralize any cationsformed as the protecting groups attached to the side chain functionalgroups of the assembled peptide chain are removed.

It is well known to those skilled in the art that proteins or peptidesthat are produced by any of the above methods can be further chemicallymodified to enhance the protein half-life in the circulation. By way ofnon-limiting example, polyethylene glycol (PEG) residues can be attachedto the penetrating peptides of the invention. Conjugating biomoleculeswith PEG, a process known as pegylation, is an established method forincreasing the circulating half-life of proteins. Polyethylene glycolsare nontoxic water-soluble polymers that, because of their largehydrodynamic volume, create a shield around the pegylated molecule,thereby protecting it from renal clearance, enzymatic degradation, aswell as recognition by cells of the immune system.

Agent-specific pegylation methods have been used in recent years toproduce pegylated molecules (e.g., drugs, proteins, agents, enzymes,etc.) that have biological activity that is the same as, or greaterthan, that of the “parent” molecule. These agents have distinct in vivopharmacokinetic and pharmacodynamic properties, as exemplified by theself-regulated clearance of pegfilgrastim, the prolonged absorptionhalf-life of pegylated interferon alpha-2a. Pegylated molecules havedosing schedules that are more convenient and more acceptable topatients, which can have a beneficial effect on the quality of life ofpatients. (See e.g., Yowell S. L. et aL, Cancer Treat Rev 28 Suppl.A:3-6 (April 2002)).

The invention also includes a method of contacting biological barrierwith a penetrating module or peptide in an amount sufficient to enableefficient penetration between the cells. The module or peptide can beprovided in vitro, ex vivo, or in vivo. Furthermore, the penetratingpeptide according to this invention may be capable of potentializing thebiological activity of the coupled substance. Therefore, another purposeof this invention is a method of using penetrating peptides to increasethe biological activity of the effector to which it is coupled.

In addition to the peptide-effector modules or peptides andpeptide-molecular vessel-effector modules, the invention also provides apharmaceutically acceptable base or acid addition salt, hydrate, ester,solvate, prodrug, metabolite, stereoisomer, or mixture thereof Theinvention also includes pharmaceutical formulations comprising apeptide-effector “module” or peptide-molecular vessel-effector module inassociation with a pharmaceutically acceptable carrier, diluent,protease inhibitor, surface active agent, or excipient.

Salts encompassed within the term “pharmaceutically acceptable salts”refer to non-toxic salts of the compounds of this invention which aregenerally prepared by reacting the free base with a suitable organic orinorganic acid to produce “pharmaceutically-acceptable acid additionsalts” of the compounds described herein. These compounds retain thebiological effectiveness and properties of the free bases.Representative of such salts are the water-soluble and water-insolublesalts, such as the acetate, amsonate(4,4-diaminostilbene-2,2′-disulfonate), benzenesulfonate, benzonate,bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calciumedetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate,embonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts.

According to the methods of the invention, a human patient can betreated with a pharmacologically effective amount of a peptide ormodule. The term “pharmacologically effective amount” means that amountof a drug or pharmaceutical agent (the effector) that will elicit thebiological or medical response of a tissue, system, animal or human thatis being sought by a researcher or clinician.

The invention also includes pharmaceutical compositions suitable forintroducing an effector of interest across a biological barrier. Thecompositions are preferably suitable for internal use and include aneffective amount of a pharmacologically active compound of theinvention, alone or in combination, with one or more pharmaceuticallyacceptable carriers. The compounds are especially useful in that theyhave very low, if any, toxicity.

Preferred pharmaceutical compositions are tablets and gelatin capsulescomprising the active ingredient together with a) diluents, e.g.lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and/orglycine; b) protease inhibitors; c) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt, poloxamer and/orpolyethyleneglycol; for tablets also d) binders, e.g., magnesiumaluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired e)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or f) absorbents, colorants, flavors andsweeteners. Injectable compositions are preferably aqueous isotonicsolutions or suspensions, and suppositories are advantageously preparedfrom fatty emulsions or suspensions. The compositions may be sterilizedand/or contain adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure and/or buffers. In addition, they may also contain othertherapeutically valuable substances. The compositions are preparedaccording to conventional mixing, granulating or coating methods,respectively, and contain about 0.01 to 75%, preferably about 0.1 to10%, of the active ingredient.

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for therapeutic agents.These methods include systemic administration such as intravenous, orlocal administration such as oral, bucal, anal, bronchial, nasal,parenteral, transdermal, subcutaneous, or topical administration modes.

Depending on the intended mode of administration, the compositions maybe in solid, semi-solid or liquid dosage form, such as, for example,injectables, tablets, suppositories, pills, time-release capsules,powders, liquids, suspensions, aerosol or the like, preferably in unitdosages. The compositions will include an effective amount of activecompound or the pharmaceutically acceptable salt thereof, and inaddition, may also include any conventional pharmaceutical excipientsand other medicinal or pharmaceutical drugs or agents, carriers,adjuvants, diluents, protease inhibitors, etc., as are customarily usedin the pharmaceutical sciences.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like may beused. The active compound defmed above, may be also formulated assuppositories using for example, polyalkylene glycols, for example;propylene glycol, as the carrier.

Liquid, particularly injectable compositions can, for example, beprepared by dissolving, dispersing, etc. The active compound isdissolved in or mixed with a pharmaceutically pure solvent such as, forexample, water, saline, aqueous dextrose, glycerol, ethanol, and thelike, to thereby form the injectable solution or suspension.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances such asfor example, sodium acetate, triethanolamine oleate, etc.

Parental injectable administration is generally used for subcutaneous,intramuscular or intravenous injections and infusions. Injectables canbe prepared in conventional forms, either as liquid solutions orsuspensions or solid forms suitable for dissolving in liquid prior toinjection.

One approach for parenteral administration employs the implantation of aslow-release or sustained-released system, which assures that a constantlevel of dosage is maintained, according to U.S. Pat. No. 3,710,795,incorporated herein by reference.

Those skilled in the art will recognize that the penetrating modules orpeptides of the instant invention can be used as an oral vaccine. Such avaccine may comprise a penetrating peptide coupled with a desiredantigenic sequence, including, but not limited to, the PA antigen ofAnthrax. This fusion protein is then orally administered to a subject inneed of vaccination.

An “antigen” is a molecule or a portion of a molecule capable ofstimulating an immune response, which is additionally capable ofinducing an animal or human to produce antibody capable of binding to anepitope of that antigen. An “epitope” is that portion of any moleculecapable of being recognized by and bound by a major histocompatabilitycomplex (“MHC”) molecule and recognized by a T cell or bound by anantibody. A typical antigen can have one or more than one epitope. Thespecific recognition indicates that the antigen will react, in a highlyselective manner, with its corresponding MHC and T cell, or antibody andnot with the multitude of other antibodies which can be evoked by otherantigens.

A peptide is “immunologically reactive” with a T cell or antibody whenit binds to an MHC and is recognized by a T cell or binds to an antibodydue to recognition (or the precise fit) of a specific epitope containedwithin the peptide. Immunological reactivity can be determined bymeasuring T cell response in vitro or by antibody binding, moreparticularly by the kinetics of antibody binding, or by competition inbinding using known peptides containing an epitope against which theantibody or T cell response is directed as competitors.

Techniques used to determine whether a peptide is immunologicallyreactive with a T cell or with an antibody are known in the art.Peptides can be screened for efficacy by in vitro and in vivo assays.Such assays employ immunization of an animal, e.g., a mouse, a rabbit ora primate, with the peptide, and evaluation of the resulting antibodytiters.

Also included within the invention are vaccines that can elicit theproduction of secretory antibodies (IgA) against the correspondingantigen, as such antibodies serve as the first line of defense against avariety of pathogens. Oral vaccination, which has the advantages ofbeing a non-invasive route of administration, is the preferred means ofimmunization for obtaining secretory antibodies.

The compounds of the present invention can be administered in such oraldosage forms as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups and emulsions. Likewise, they may also beadministered in intravenous (both bolus and infusion), intraperitoneal,subcutaneous or intramuscular form, and all using forms well known tothose of ordinary skill in the pharmaceutical arts.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic finction ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, may be provided in the form of scored tablets containing 0.005,0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0,50.0, 100.0, 250.0, 500.0 or 1000.0 mg of active ingredient.

Compounds of the present invention may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three or four times daily. Furthermore, preferred compounds for thepresent invention can be administered in bucal form via topical use ofsuitable bucal vehicles, bronchial form via suitable aerosols orinhalants, intranasal form via topical use of suitable intranasalvehicles, or via trnsdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen. Other preferred topical preparationsinclude creams, ointments, lotions, aerosol sprays and gels, wherein theconcentration of active ingredient would range from 0.1% to 15%, w/w orw/v.

The compounds herein described in detail can form the active ingredient,and are typically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, protease inhibitors, disintegrating agentsand coloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,poloxamer, polyethylene glycol, waxes and the like. Lubricants used inthese dosage forms include sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride and the like.Disintegrators include, without limitation, starch, methylcellulose,agar, bentonite, xanthan gum and the like.

The compounds of the present invention can also be administered in theform of liposome delivery systems, such as small unilamellar vesicles,large unilamellar vesicles and multilamellar vesicles. Liposomes can beformed from a variety of phospholipids, containing cholesterol,stearylamine or phosphatidylcholines. In some embodiments, a film oflipid components is hydrated with an aqueous solution of drug to a formlipid layer encapsulating the drug, as described in U.S. Pat. No.5,262,564.

The compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

Any of the above pharmaceutical compositions may contain 0.01-99%,preferably 0.1-10% of the active compounds as active ingredients.

The following EXAMPLES are presented in order to more fully illustratethe preferred embodiments of the invention. These EXAMPLES should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLES Example 1 Demonstration of the Efficacy of the PenetratingModule to Enable the Translocation of a Peptide Through an EpithelialBarrier

The tested peptide, representing a penetrating peptide and aradio-labeled probe in a contiguous construct of SEQ ID NO 3 and SEQ IDNO 17, hereby named IBW 002 (Novetide, lot 40139-45, sequence:Ac-N-Y-Y-D-I-T-L-A-L-A-G-I-C-Q-S-A-R-L-V-Q-Q-L-A-G-G-G-K-G-G-K-NH₂ (SEQID NO:22), was ¹²⁵I-labeled using the Bolton Hunter Reagent (Biochem. J.133:529-39 (1973)). The labeled peptide was loaded on a Sephadex G10column, washed with phosphate buffer and eluted with 3M Tiocyanate inDDW+50% n-butanol. The peak, containing 6 fractions, was kept and usedfor in vivo experiments.

The test sample was prepared as shown in Table 2 TABLE 2 ¹²⁵I-IBW-002sample  26 ul PE 6,200  20 ul Aprotinin 100 ul PB 849 ul NAC (stock 9.8mg/ml)  5 ulIn vivo Experimental Procedure:

Four male BALB/c mice, 9-10 weeks old, were deprived of food, 18 hoursprior to the experiment.

The mice were anesthetized by i.p. injection of 0.05 ml of a mixture of0.15 ml xylazine+0.85 ml of ketamin. A 2 cm long incision was made alongthe center of the abdomen, through the skin and abdominal wall. Anintestine loop was gently pulled out through the incision and placed onwet gauze beside the animal. The loop remained intact through the entireprocedure and was kept wet during the whole time. The tested compoundwas injected into the loop, 0.2 ml/ mouse, containing around 200,000cpm, using a 26G needle. Fifteen minutes post injection the tip of thetail was cut and a 50 μl blood sample was drawn into a glass capillary.This was repeated 30 and 60 minutes post injection. The animals weresubsequently sacrificed. The following organs were removed: epididymalfat, kidneys, liver, lungs and brain. Radioactivity in each organ and inblood samples was counted. A 5 μl sample of the injected solution wascounted to determine the total injected cpm per mouse.

Results:

The total radioactive labeling injected per mouse was 224,160 cpm. Thecpm recovered from each tissue is summarized in Table 3. Total cpm inthe blood was calculated based on the 50 μl blood sample drawn out atdifferent time points post injection, in the assumption that total bloodvolume of each mouse is 2 ml. TABLE 3 mouse #1 mouse #2 mouse #3 mouse#4 mean ±SEM blood 15′ 37,080 68,960 62,280 80,280 62,150 9,145 blood30′ 36,120 60,680 52,320 70,840 54,990 7,342 blood 60′ 29,720 47,80044,000 59,720 45,310 6,182 Epididymal 547 869 973 1,174 891 131 fatkidney 4,068 6,614 6,400 7,650 6,183 756 liver 6,102 9,709 8,718 11,6749,051 1,159 lungs 865 1,343 1,349 1,806 1,341 192 brain 138 190 202 301208 34 recovered 41,440 66,525 61,642 82,325 62,983 8,429 cpm/mouse18.49 29.68 27.50 36.73 28.10 3.76 recovered/ injected cpm (%)

The percent of ¹²⁵I-IBW-002 in the blood relative to the total injected¹²⁵I-IBW-002, at the 3 different time points, is presented in Table 4.

As can be seen in Table 4, the relative amount of ¹²⁵I-IBW-002 in theblood peaked already at 15 min. post-intestinal administration anddecreased with time. This probably indicates peptide distribution fromthe blood to various organs. TABLE 4 mouse mouse mouse mouse #1 #2 #3 #4mean ±SEM blood/injected 16.54 30.76 27.78 35.81 27.73 4.08 cpm (%), 15′blood/injected 16.11 27.07 23.34 31.60 24.53 3.28 cpm (%), 30′blood/injected 13.26 21.32 19.63 26.64 20.21 2.76 cpm (%), 60′

In similar experiments, a control ¹²⁵I-labeled peptide (SEQ ID NO:17),lacking the penetrating peptide-sequence, was injected into a mouseintestinal loop. The average amount of the control peptide recoveredfrom the blood and various organs was only about ⅕ of that obtained withthe full conjugate of the IBW-002 peptide, although this control peptideis about ¼ in its size as compared with IBW-002.

Example 2 Development of Optimal Formulation.

To reach an improved formulation, a number of compounds were tested inexperiments similar to that described in Example 1, with the followingchange: urine was collected and the measured radioactivity was added tothat of the recovered tissues.

a. Non Ionic Detergents (Preferably Poloxamers) TABLE 5 Initialformulation with Pluronic PE 6,200 Peptide sample (radiolabeled) 10 ulPE 6,200 20 ul Aprotinin 100 ul  PB  5 ul NAC (9.8 mg/ml)  5 ul

The total radioactive labeling injected per mouse was 125,320 cpm. Thecpm recovered from each tissue is summarized in Table 6. Total cpm inthe blood was calculated based on the 50 μl blood sample drawn out atdifferent time points post injection, in the assumption that total bloodvolume of each mouse is 2 ml. TABLE 6 mouse #1 mouse #2 mean ±SEM blood15′ 19,920 19,560 19,740 180 blood 30′ 18,800 15,880 17,340 1,460 blood60′ 14,560 10,960 12,760 1,800 epididimal 321 288 305 17 fat kidney2,564 3,799 3,182 618 liver 2,994 2,786 2,890 104 lungs 476 377 427 50brain 95 110 103 8 urine 14,749 21,747 18,248 3,499 recovered 35,75940,067 37,913 2,154 cpm/mouse recovered 28.53 31.97 30.25 1.72 cpm/injected cpm (%)

The percent of ¹²⁵I-IBW-002 in the blood relative to the total injected¹²⁵I-IBW-002, at the 3 different time points, is presented in Table 7TABLE 7 mouse #1 mouse #2 mean ±SEM blood/injected cpm (%), 15′ 15.9015.61 15.75 0.14 blood/injected cpm (%), 30′ 15.00 12.67 13.84 1.17blood/injected cpm (%), 60′ 11.62 8.75 10.18 1.44

Calculated total recovery was 30.25%.

b. Bile Salts (Preferably Taurodeoxycholic Acid) TABLE 8 Peptide sample(radiolabeled) 10 ul PE 6,200 20 ul Aprotinin 100 ul PB 802.5 ul NAC 9.8mg/ml 5 ul Taurodeoxycholic acid (8% in DDW) 62.5 ul

The total radioactive labeling injected per mouse was 195,900 cpm. Thecpm recovered from each tissue is summarized in Table 9. Total cpm inthe blood was calculated based on the 50 μl blood sample drawn out atdifferent time points post injection, in the assumption that total bloodvolume of each mouse is 2 ml. TABLE 9 mouse #1 mouse #2 mouse #3 mouse#4 mean ±SEM blood 15′ 40,600 39,440 51,920 36,200 42,040 3,422 blood30′ 37,440 31,720 48,440 34,720 38,080 3,646 blood 60′ 33,880 23,08040,840 30,200 32,000 3,702 epididimal fat 1,081 805 903 846 909 61kidney 9,513 7,878 10,661 8,929 9,245 581 liver 8,194 5,154 9,615 8,7087,918 967 lungs 1,048 636 1,221 1,303 1,052 149 brain 206 176 284 173210 26 urine 39,992 30,651 25,445 40,361 34,112 3,660 recovered 93,91468,380 89,969 90,520 85,446 5,782 cpm/mouse 47.94 34.91 45.42 46.2143.62 2.95 recovered cpm/ injected cpm (%)

The percent of ¹²⁵I-IBW-002 in the blood relative to the total injected¹²⁵I-IBW-002, at the 3 different time points, is presented in Table 10.TABLE 10 mouse mouse mouse mouse #1 #2 #3 #4 mean ±SEM blood/injected20.72 20.13 26.50 18.48 21.46 1.75 cpm (%), 15′ blood/injected 19.1116.19 24.73 17.72 19.44 1.86 cpm (%), 30′ blood/injected 17.29 11.7820.85 15.42 16.33 1.89 cpm (%), 60′

Calculated total recovery was 43.62%.

c. Pluronic F-68 as a Preferred Poloxamer TABLE 11 Peptide sample(radiolabeled) 18 ul Pluronic F-68 (2.72% in PB) 735 ul Aprotinin 100 ulPB 80 ul NAC (9.8 mg/ml) 5 ul Taurodeoxycholic acid (8% in DDW) 62.5 ul

The total radioactive labeling injected per mouse was 143,000 cpm. Thecpm recovered from each tissue is summarized in Table 12. Total cpm inthe blood was calculated based on the 50 μl blood sample drawn out atdifferent time points post injection, in the assumption that total bloodvolume of each mouse is 2 ml. TABLE 12 mouse #1 mouse #2 mouse #3 mouse#4 mean ±SEM blood 5′ 29,400 29,120 34,240 32,920 31,420 1,277 blood 10′26,680 32,320 28,200 27,760 28,740 1,235 blood 20′ 20,280 30,960 27,24021,800 25,070 2,467 blood 60′ 11,880 22,840 16,280 14,440 16,360 2,341epididimal fat 375 591 462 465 473 44 kidney 2,397 4,407 5,018 5,7724,399 723 liver 3,516 6,546 6,561 4,807 5,358 739 lungs 393 643 582 533538 53 brain 76 106 94 74 88 8 urine 59,442 32,158 35,831 53,271 45,1766,620 recovered 78,079 67,291 64,828 79,362 72,390 3,699 cpm/mouse 54.6047.06 45.33 55.50 50.62 2.59 recovered cpm/ injected cpm (%)

The percent of ¹²⁵I-IBW-002 in the blood relative to the total injected¹²⁵I-IBW-002, at the four different time points, is presented in Table13. TABLE 13 mouse mouse mouse mouse #1 #2 #3 #4 mean ±SEMblood/injected 20.56 20.36 23.94 23.02 21.97 0.89 cpm (%), 5′blood/injected 18.66 22.60 19.72 19.41 20.10 0.86 cpm (%), 10′blood/injected 14.18 21.65 19.05 15.24 17.53 1.73 cpm (%), 20′blood/injected 8.31 15.97 11.38 10.10 11.44 1.64 cpm (%), 60′

Calculated total recovery was 50.62%. TABLE 14 Summary of formulationresults regarding the added detergents Formulation Recovery (%) 2% PE6,200 30.25 2% PE 6,200 + 0.5% Taurodeoxycholic acid 43.62 2% PluronicF-68 + 0.5% Taurodeoxycholic acid 50.62

Example 3 Relative Potency of Penetrating Peptides as Tested in Mice.

TABLE 15 SEQ Relative Peptide's ID potency name NO. Sequence (%) IBW-00222 AcNYYDITLALAGICQSARLVQQLAGGGK 100 GGKNH₂ IBW-002V2 36AcMYYDITLALAGICQSARLVQQLAGGGK 80 GGKNH₂ IBW-002V3 37AcMYDITLALAGICQSARIVQQLAGGKGG 77 KNH₂ IBW-006 33AcNYHDIVLALAGVCQSARLVHQLAGGKG 77 GKNH₂ IBW-002V1 35AcMNYYDITLALAGICQSARLVQQLAGGG 73 KGGKNH₂ IBW-007 34AcNYFLVNLAFAEASMAAFNTVVNFGGKG 66 GKNH₂ IBW-004 31AcNYFIVNLALADLCMAAFNAAFNFGGGK 63 GGKNH₂ IBW-005 32AcMRNLTRTSLLLAGLCTAAQMVFVGGGK 51 GGKNh₂ IBW-003 30AcNLPPIVLAVIGICAAVFLLQQYVGGGK 37 GGKNH₂

Example 4 Conditionally-activated Effectors: Inhibition of ExcessiveBlood Coagulation

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to a coagulation inhibitory protein (e.g., hirudin, hirulog,Protein C, Protein C activator or Protein S) or a thrombolysisactivating protein (ie., tPA) through the sequence IEGR (SEQ ID NO: 16),a cleavage site recognized by factor Xa.

At sites of excessive blood coagulation, the linker sequence will becleaved by the abundant factor Xa, thereby releasing the coupledcoagulation inhibitory protein. As a result, the coagulation processwill be attenuated. Inhibition of excessive or pathological coagulationcan be assessed locally by reduction in thrombus size, morphology, andpropagation along the vasculature.

Consequently, a reduction of ischemic damage can be demonstrated viahistological and functional extent of an infarct. Systemic effects canbe assessed by lengthened Prothromin Time (PT) and PartialThromboplastin Time (PTT), and reduced levels of fibrinogen degradationproducts and D-dimers (FDPs).

Example 5 Conditionally-activated Effectors: Inhibition of PathologicInflammatory Processes

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to a complement inhibitory protein (e.g., a Clq inhibitor)through a cleavage site that is recognized by complement cascadeproteins (e.g., C3a).

At instances of pathological complement activation, such as excessiveinflammatory processes, the complement inhibitor will be released.Inhibition of the complement cascade can be assessed directly by areduction in CH50, or individual C3 and C4 activity. Overall modulationof inflammation may be determined by a reduction in levels of acutephase proteins, e.g., C-reactive protein (CRP), or a reduction inelevated sedimentation rate (ESR).

Example 6 Utilization of the Penetrating Module for Oral Vaccination

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to a desired antigenic sequence. For example, a fusion proteincomposed of the penetrating module coupled to the PA antigen of Anthrax.Such a fusion protein can be administered to a subject in need ofvaccination.

This method allows simple and rapid vaccination of large populations inneed thereof. Another advantage of this method is the production of hightiters of IgA antibodies and the subsequent presence of IgA antibodiesin the epithelial mucosa, which are the sites of exposure to antigens.

Efficacy of vaccination can be demonstrated by the measurement ofspecific antibody titers, especially for IgA, as well as the measurementof immunological response to stimulation, such as for example, via acutaneous hyerpsensitivity reaction in response to subcutaneousadministration of antigen.

Example 7 Utilization of the Penetrating Module for Bulk Translocation

By covalently attaching stearyl residues to the free amino groups of thetwo lysines at the C-terminus of SEQ ID NO:22, a hydrophobizedpenetrating peptide is produced. This hydrophobized penetrating peptideis then incorporated into the interface of taxol-containingmicroparticles composed of triglyceride medium.

This method allows for the translocation of highly hydrophobic drugs,such as taxol, from the intestine into the bloodstream. Because suchdrugs cannot be otherwise absorbed from the intestine, they aretypically administered to patients via invasive means.

The effects of taxol-containing microparticles can be demonstrated by areduction in tumor size, metastases or other tumor-related markers.

Example 8 Recombinant Human Insulin (rh-Insulin) Delivery Across MucosalEpithelia via a Fusion Construct

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to rh-Insulin in one of two ways:

1. through a cleavage site: (SEQ ID NO:18)NYYDITLALAGICQSARLVQQLA-GG-IEGR- rh-Insulin

2. without a cleavage site: (SEQ ID NO:19) NYYDITLALAGICQSARLVQQLA-GG-rh-Insulin.

The resulting compound is dissolved in a 2% pluronic PE 6200 aqueoussolution. Two hundred μL of this solution, (or vehicle alone), isinjected into an intestinal loop of a mouse and blood glucose levels aresubsequently measured.

Blood glucose levels decrease in relation to the amount of insulinabsorbed from the intestine into the bloodstream (i.e., in an amountthat correlates to the amount of insulin absorbed). Thus, this drugdelivery system can replace the need for insulin injections, therebyproviding an efficient, safe and convenient route of administration fordiabetes patients.

Example 9 Recombinant Human Insulin (rh-Insulin) Delivery Across MucosalEpithelia via a Molecular Vessel Fusion Construct

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to a ligand binding domain of the insulin receptor (orminireceptor) which, in turn, is bound to rh-Insulin as follows:

NYYDITLALAGICQSARLVQQLA-GG- linearized insulin receptor (SEQ ID NO:23)(see Kristensen, et al., J. Biol. Chem., 274(52):37351-56 (1999)) plusan equimolar amount of rh-insulin.

The resulting complex is dissolved in a 2% pluronic PE 6200 aqueoussolution. Two hundred μL of the above dissolved complex, (or vehiclealone), is injected into an intestinal loop of a mouse and blood glucoselevels are subsequently measured.

Blood glucose levels decrease in relation to the amount of insulinabsorbed from the intestine into the bloodstream (ie., in an amount thatcorrelates to the amount of insulin absorbed). Thus, this drug deliverysystem can replace the need for insulin injections, thereby providing anefficient, safe and convenient route of administration for diabetespatients.

Example 10 Low Molecular Weight Heparin Delivery Across MucosalEpithelia

SEQ ID NO:3 (or any other sequence from SEQ ID NO:1-15, 24-29) iscoupled to heparin for example, through the free amino group of an extralysine added at the C-terminus, in one of two ways:

1. through a cleavage site: (SEQ ID NO:20)NYYDITLALAGICQSARLVQQLA-GG-IEGR-K- heparin

2. without a cleavage site: (SEQ ID NO:21) NYYDITLALAGICQSARLVQQLA-GG-K-heparin.

The resulting compound is dissolved in a 2% pluronic PE 6200 aqueoussolution. Two hundred μL of this solution, (or vehicle alone), isinjected into an intestinal loop of a mouse. Partial Thrombin Time (PTT)values are subsequently measured.

Partial Thrombin Time (PTT) values decrease in relation to the amount ofheparin absorbed from the intestine loop into the bloodstream (ie., inan amount that correlates to the amount of insulin absorbed). Therefore,this drug delivery system will replace the use of heparin injections.

OTHER EMBODIMENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique methods oftranslocation across epithelial and endothelial barriers have beendescribed. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims that follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of the particular type of tissue, or the particulareffector to be translocated is believed to be a matter of routine for aperson of ordinary skill in the art with knowledge of the embodimentsdescribed herein.

1. A penetrating peptide comprising at least one amino acid sequenceselected from the group consisting of: a) (BX)₄Z(BX)₂ZXB; b)ZBXB₂XBXB₂XBX₃BXB₂X₂B₂; c) ZBZX₂B₄XB₃ZXB₄Z₂B₂; d) ZB₉XBX₂B₂ZBXZBX₂; e)BZB₈XB₉X₂ZXB; f) B₂ZXZB₅XB₂XB₂X₂BZXB₂; g) XB₉XBXB₆X₃B; h)X₂B₃XB₄ZBXB₄XB_(n)XB; i) XB₂XZBXZB₂ZXBX₃BZXBX₃B; j)BZXBXZX₂B₄XBX₂B₂XB₄X₂; k) BZXBXZX₂B₄XBX₂B₂XB₄; l) B₂XZ₂XB₄XBX₂B₅X₂B₂; m)B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; n)B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; o) X₃ZB₆XBX₃BZB₂X₂B₂; and p) atleast 12 contiguous amino acids of any of peptides a) through o) whereinq is 0 or 1; m is 1 or 2; n is 2 or 3; t is 1 or 2 or 3; and X is anyamino acid; B is a hydrophobic amino acid; and Z is a charged aminoacid; wherein said penetrating peptide is capable of translocatingacross a biological barrier.
 2. The penetrating peptide of claim 1,wherein the amino acid sequence is selected from the group consistingof: a) SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,24, 25, 26, 27, 28 and 29; b) a variant of an amino acid sequenceselected from the group consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 24, 25, 26, 27, 28 and 29, wherein one ormore amino acid residues in said variant differs from the amino acidsequence of said penetrating peptide, provided that said variant differsin no more than 15% of amino acid residues from said amino acidsequence; c) a fragment of an amino acid sequence selected from thegroup consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 24, 25, 26, 27, 28 and 29; and d) a peptide comprising atleast 12 contiguous amino acids of any of the peptides selected from thegroup consisting of SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 24, 25, 26, 27, 28 and
 29. 3. The penetrating peptide ofclaim 2, wherein the fragment is at least 10 amino acids in length. 4.The penetrating peptide of claim 2, wherein the amino acid sequence ofsaid variant comprises a conservative amino acid substitution.
 5. Thepenetrating peptide of claim 2, wherein the amino acid sequence of saidvariant comprises a non-conservative amino acid substitution.
 6. Thepenetrating peptide of claim 1, wherein the amino acid sequence is SEQID NO: 3 or at least 12 contiguous amino acids thereof.
 7. Thepenetrating peptide of claim 1, wherein the amino acid sequence is SEQID NO: 8 or at least 12 contiguous amino acids thereof.
 8. Thepenetrating peptide of claim 1, wherein the amino acid sequence is SEQID NO: 9 or at least 12 contiguous amino acids thereof.
 9. Thepenetrating peptide of claim 1, wherein the amino acid sequence is SEQID NO: 12 or at least 12 contiguous amino acids thereof.
 10. Thepenetrating peptide of claim 1, wherein the amino acid sequence is SEQID NO: 24 or at least 12 contiguous amino acids thereof.
 11. Thepenetrating peptide of claim 1, wherein the peptide is less than 30amino acids long.
 12. The penetrating peptide of claim 1, wherein thepeptide is less than 25 amino acids long.
 13. The penetrating peptide ofclaim 1, wherein the peptide is less than 20 amino acids long.
 14. Apenetrating peptide comprising at least one amino acid sequence selectedfrom the group consisting of: a) (BX)₄Z(BX)₂ZXB; b)ZBXB₂XBXB₂XBX₃BXB₂X₂B₂; c) ZBZX₂B₄XB₃ZXB₄Z₂B₂; d) ZB₉XBX₂B₂XBXZBX₂; e)BZB₈XB₉X₂ZXB; f) B₂ZXZB₅XB₂XB₂X₂BZXB₂; g) XB₉XBXB6X₃B; h)X₂B₃XB₄ZBXB₄XB_(n)XB; i) XB₂XZBXZB₂ZXBX₃BZXBX₃B; j)BZXBXZX₂B₄XBX₂B₂XB₄X₂; k) BZXBXZX₂B₄XBX₂B₂XB₄; l) B₂XZ₂XB4XBX₂B₅X₂B₂; m)B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; n)B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; o) X₃ZB₆XBX₃BZB₂X₂B₂; and p) atleast 12 contiguous amino acids of any of peptides a) through o) whereinq is 0 or 1; m is 1 or 2; n is 2 or 3; t is 1 or 2 or 3; and X is anyamino acid; B is a hydrophobic amino acid; and Z is a charged aminoacid; wherein said penetrating peptide is capable of translocatingacross a biological barrier and wherein the penetrating peptide iscoupled to an effector.
 15. The penetrating peptide of claim 14, whereinsaid effector is a bioactive peptide.
 16. The penetrating peptide ofclaim 15, wherein said bioactive peptide is selected from the groupconsisting of: insulin, growth hormone, a gonadotropin, a growth factor,erythropoietin, granulocyte/monocyte colony stimulating factor (GM-CSF),αMSH, enkephalin, dalargin, kyotorphin, EPO, bFGF, hirulog, a lutenizinghormone releasing hormone (LHRH) analog, and neurotrophic factors. 17.The penetrating peptide of claim 14, wherein said effector is apharmaceutically active agent.
 18. The penetrating peptide of claim 17,wherein said pharmaceutically active agent is selected from the groupconsisting of: an anticoagulant, a toxin, an antibiotic, anantipathogenic agent, an antigen, an antibody fragment, animmunomodulator, a vitamin, an enzyme, an antineoplastic agent, and atherapeutic agent.
 19. A method of translocating a penetrating peptideacross a biological barrier, wherein the penetrating peptide is thepenetrating peptide of claim
 2. 20. A method of translocating apenetrating peptide across a biological barrier, wherein the penetratingpeptide is the penetrating peptide of claim
 6. 21. A method oftranslocating a penetrating peptide across a biological barrier, whereinthe penetrating peptide is the penetrating peptide of claim
 7. 22. Amethod of translocating a penetrating peptide across a biologicalbarrier, wherein the penetrating peptide is the penetrating peptide ofclaim
 8. 23. A method of translocating a penetrating peptide across abiological barrier, wherein the penetrating peptide is the penetratingpeptide of claim
 9. 24. A method of translocating a penetrating peptideacross a biological barrier, wherein the penetrating peptide is thepenetrating peptide of claim
 10. 25. A penetrating peptide comprising atleast one penetrating peptide having an amino acid sequence selectedfrom the group consisting of: a) (BX)₄Z(BX)₂ZXB; b)ZBXB₂XBXB₂XBX₃BXB₂X₂B₂; c) ZBZX₂B₄XB₃ZXB₄Z₂B₂; d) ZB₉XBX₂B₂XBXZBX₂; e)BZB₈XB₉X₂ZXB; f) B₂ZXZB₅XB₂XB₂X₂BZXB₂; g) XB₉XBXB₆X₃B; h)X₂B₃XB₄ZBXB₄XB_(n)XB; i) XB₂XZBXZB₂ZXBX₃BZXBX₃B; j)BZXBXZX₂B₄XBX₂B₂XB₄X₂; k) BZXBXZX₂B₄XBX₂B₂XB₄; l) B₂XZ₂XB₄XBX₂B₅X₂B₂; m)B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; n)B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; o) X₃ZB₆XBX₃BZB₂X₂B₂; and p) atleast 12 contiguous amino acids of any of peptides a) through o) whereinq is 0 or 1; m is 1 or 2; n is 2 or 3; t is 1 or 2 or 3; and X is anyamino acid; B is a hydrophobic amino acid; and Z is a charged aminoacid; wherein said penetrating peptide is capable of translocatingacross a biological barrier and wherein said penetrating peptide isfused to an effector.
 26. The penetrating peptide as in any one ofclaims 1, 14, and 25, wherein translocation across a biological barrieroccurs within a tissue selected from the group consisting of: epithelialcells and endothelial cells.
 27. The penetrating peptide as in any oneof claims 1, 14 and 25, wherein said biological barrier is selected fromthe group consisting of: tight junctions and the plasma membrane. 28.The penetrating peptide of claim 25, wherein the effector is selectedfrom the group consisting of: a bioactive peptide and a pharmaceuticallyactive agent.
 29. The penetrating peptide of claim 28, wherein saidbioactive peptide is selected from the group consisting of: insulin,growth hormone, a gonadotropin, a growth factor, erythropoietin, GM-CSF,αMSH, enkephalin, dalargin, kyotorphin, EPO, bFGF, hirulog, an LHRHanalog, and neurotrophic factors.
 30. The penetrating peptide of claim28, wherein said pharmaceutically active agent is selected from thegroup consisting of: an anticoagulant, a toxin, an antibiotic, anantipathogenic agent, an antigen, an antibody fragment, a vitamin, animmunomodulator, an enzyme, an antineoplastic agent, heparin,methotraxate, and a therapeutic agent.
 31. A pharmaceutical compositioncomprising a therapeutically or prophylactically effective amount of thepenetrating peptide according to claim 25, and a pharmaceuticallyacceptable carrier.
 32. The pharmaceutical composition of claim 31,wherein the composition further comprises a mixture of at least twosubstances selected from the group consisting of a non-ionic detergent,an ionic detergent, a protease inhibitor; and a reducing agent.
 33. Thepharmaceutical composition of claim 32, wherein the non-ionic detergentis a poloxamer.
 34. The pharmaceutical composition of claim 33, whereinthe poloxamer is pluronic F-68.
 35. The pharmaceutical composition ofclaim 32, wherein the ionic detergent is a bile salt.
 36. Thepharmaceutical composition of claim 35, wherein the bile salt isTaurodeoxychilate.
 37. The pharmaceutical composition of claim 32,wherein the protease inhibitor is selected from the grove consisting ofaprotonin and soya bean trypsin inhibitor.
 38. The pharmaceuticalcomposition of claim 32, wherein the reducing agent is NAC.
 39. A methodof producing a penetrating peptide comprising the penetrating peptide ofclaim 1 and an effector, said method comprising coupling said effectorto said penetrating peptide.
 40. The method of claim 39, wherein thecoupling of said effector is achieved by a covalent bond.
 41. The methodof claim 40, wherein said covalent bond is a peptide bond.
 42. Themethod of claim 40, wherein the covalent bond is achieved by a homo- ora hetero-finctional bridging reagent.
 43. The method of claim 42,wherein the bridging reagent is a succinimidyl-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC)-type reagent.
 44. The method of claim40, wherein the covalent bond is achieved by a peptide linker.
 45. Themethod of claim 44, wherein the peptide linker has the sequence of SEQID NO: 16 or SEQ ID NO:17.
 46. The method of claim 44, wherein thepeptide linker can be cleaved by an enzyme.
 47. The method of claim 46,wherein the peptide linker is designed to be cleaved by an enzymeconditionally activated under a certain physiological state and whereinthe released effector favorably influences that physiological state. 48.The method of claim 39, wherein the coupling of said effector isachieved by a non-covalent bond.
 49. The method of claim 48, wherein thenon-covalent bond is achieved by an attachment of a hydrophobic moietyto the penetrating peptide, wherein the hydrophobic moiety enablespenetrating module to be incorporated at the interface of a hydrophobicvesicle in which the effector is contained.
 50. The method of claim 48,wherein the non-covalent bond is the result of a biotin-avidin orbiotin-streptavidin interaction.
 51. A method of translocating aneffector across a biological barrier, said method comprising: a)coupling said effector to the penetrating peptide of claim 1 to form apenetrating module; and b) introducing said penetrating module to thebiological barrier.
 52. A method of oral vaccination, wherein the methodcomprises administering to subject the penetrating peptide of claim 1coupled with a desirable antigen.
 53. A kit comprising, in one or morecontainers, a therapeutically or prophylactically effective amount ofthe pharmaceutical composition of claim
 31. 54. A method of treating orpreventing a disease or pathological condition, said method comprisingadministering to a subject in which such treatment or prevention isdesired, the pharmaceutical composition of claim 31, in an amountsufficient to treat or prevent said disease or said pathologicalcondition in said subject.
 55. The method of claim 54, wherein saiddisease or said pathological condition is selected from a groupconsisting of endocrine disorders, diabetes, infertility, hormonedeficiencies, osteoporosis, neurodegenerative disorders, Alzheimer'sdisease, Parkinson's disease, Huntington's disease, cardiovasculardisorders, atherosclerosis, hypercoagulable states, hypocoagulablestates, coronary disease, cerebrovascular events, metabolic disorders,obesity, vitamin deficiencies, haematological disorders, and neoplasticdisease.
 56. A method for producing the penetrating peptide of claim 25comprising a) transfecting a production cell with a vector comprising anucleic acid molecule of a fusion protein encoding said penetratingpeptide and an effector operably linked to an expression controlsequence; b) culturing said production cell under conditions that permitproduction of a fusion protein consisting of the penetrating peptide andan effector peptide; and c) isolating said fusion protein.
 57. A peptidecomprising an amino acid sequence, wherein said peptide is derived froma pathogenic bacteria, and said peptide is characterized by the abilityto penetrate biological barriers in vivo.
 58. The peptide of claim 57,wherein the peptide is derived from an integral membrane protein. 59.The peptide of claim 57, wherein the peptide is derived from a bacterialtoxin.
 60. A peptide comprising an amino acid sequence, wherein saidpeptide is derived from a nonpathogenic bacteria, and said peptide ischaracterized by the ability to penetrate biological barriers in vivo.61. The peptide of claim 60, wherein said peptide is derived from anintegral membrane protein.
 62. The peptide of claim 60, wherein thepeptide is derived from an extracellular protein.
 63. A peptidecomprising an amino acid sequence, wherein said peptide is derived froma human neurokinin receptor, and said peptide is characterized by theability to penetrate biological barriers in vivo.
 64. A penetratingmodule comprising at least one penetrating peptide having an amino acidsequence selected from the group consisting of: a) (BX)₄Z(BX)₂ZXB; b)ZBXB₂XBXB₂XBX₃BXB₂X₂B₂; c) ZBZX₂B₄XB₃ZXB₄Z₂B₂; d) ZB₉XBX₂B₂XBXZBX₂; e)BZB₈XB₉X₂ZXB; f) B₂ZXZB₅XB₂XB₂X₂BZXB₂; g) XB₉XBXB₆X₃B; h)X₂B₃XB₄ZBXB₄XB_(n)XB; i) XB₂XZBXZB₂ZXBX₃BZXBX₃B; j)BZXBXZX₂B₄XBX₂B₂XB₄X₂; k) BZXBXZX₂B₄XBX₂B₂XB₄; l) B₂XZ₂XB₄XBX₂B₅X₂B₂; m)B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; n)B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂; o) X₃ZB₆XBX₃BZB₂X₂B₂; and p) atleast 12 contiguous amino acids of any of peptides a) through o) whereinq is 0 or 1; m is 1 or 2; n is 2 or 3; t is 1 or 2 or 3; and X is anyamino acid; B is a hydrophobic amino acid; and Z is a charged aminoacid; wherein said penetrating module is capable of translocating acrossa biological barrier and wherein said penetrating peptide is fused tomolecular vessel, wherein said molecular vessel encloses an effector.65. The penetrating module of claim 64, wherein the effector is selectedfrom the group consisting of: a bioactive peptide and a pharmaceuticallyactive agent.
 66. The penetrating module of claim 65, wherein saidbioactive peptide is selected from the group consisting of: insulin,growth hormone, a gonadotropin, a growth factor, erythropoietin, GM-CSF,αMSH, enkephalin, dalargin, kyotorphin, EPO, bFGF, hirulog, an LHRHanalog, and neurotrophic factors.
 67. The penetrating module of claim65, wherein said pharmaceutically active agent is selected from thegroup consisting of: an anticoagulant, a toxin, an antibiotic, anantipathogenic agent, an antigen, an antibody fragment, a vitamin, animmunomodulator, an enzyme, an antineoplastic agent, heparin,methotraxate, and a therapeutic agent.
 68. The penetrating module ofclaim 64, wherein the molecular vessel is selected from the groupconsisting of a soluble receptor, a minireceptor, and a binding protein.69. The penetrating module of claim 68, wherein the soluble receptor isa soluble insulin receptor.
 70. The penetrating module of claim 69,wherein the effector is insulin.
 71. The penetrating module of claim 68,wherein the minireceptor is the ligand-binding domain of the insulinreceptor.
 72. The penetrating module of claim 71, wherein the effectoris insulin.
 73. The penetrating module of claim 68, wherein the bindingprotein is Intrinsic factor.
 74. The penetrating module of claim 73,wherein the effector is vitamin B12.
 75. A pharmaceutical compositioncomprising a therapeutically or prophylactically effective amount of thepenetrating module according to claim 64, and a pharmaceuticallyacceptable carrier.
 76. A method for producing the penetrating peptideof claim 25 comprising using solid-phase synthesis of the peptide. 77.The penetrating peptide of claim 15, further comprising a chemicalmodification.
 78. The penetrating peptide of claim 77, wherein saidbioactive peptide is selected from the group consisting of: insulin,growth hormone, a gonadotropin, a growth factor, erythropoietin,granulocytelmonocyte colony stimulating factor (GM-CSF), αMSH,enkephalin, dalargin, kyotorphin, EPO, bFGF, hirulog, a lutenizinghormone releasing hormone (LHRH) analog, and neurotrophic factors. 79.The method of claim 56, wherein the fusion protein is further chemicallymodified.
 80. The method of claim 79, wherein the chemical modificationcomprises the attachment of one or more polyethylene glycol residues tothe fusion protein.